The main therapeutic challenge in the treatment of acute myeloid leukemias (AML) is the development of strategies aimed at overcoming resistance to chemotherapy and in particular to treat relapsed patients successfully. The factors regulating the survival of resistant leukemic cells are not known and may be different from those operational at diagnosis. This project is designed to identify roadblocks of apoptosis in resistant leukemia cells and to target already identified mechanisms of apoptotic resistance. We and others have analyzed the expression of pro- and anti-apoptotic genes in newly diagnosed AML. Increased levels of Bcl-2 were found to be associated with poor prognosis. This led us to conduct in vitro studies demonstrating that ara-C and anthracycline cytotoxicity in AML was markedly enhanced by Bcl-2 antisense (AS) oligonucleotides, even in the presence of other anti-apoptotic proteins, a funding that will now be tested in clinical trials (Core A). Of particular importance is the finding that Bcl-2 is expressed in early leukemic, but not in early normal progenitor cells. Furthermore, the group of inhibitor- of-apoptosis proteins (IAPs), identified recently as inactivating caspases downstream from Bcl-2, was examined in AML: anti-apoptotic XIAP and survivin are expressed in AML cell lines and primary AML and are dramatically up-regulated by cytokines. Furthermore, high levels of XIAP are associated with poor survival in AML, and increased levels were found in cells surviving exposure to ara-C and Doxorubicin. Preliminary data indicate lack of XIAP and survivin expression in early normal progenitors (CD34+38-) suggesting XIAP and survivin is differentially expressed potential therapeutic targets in AML. We now propose to further investigate the anti-apoptotic function of XIAP and survivin using AS and retinoids as potential therapeutics for AML (Specific Aim #1). Our second therapeutic target is Bad, a pro-apoptotic Bcl-2 family member, that is rendered anti-apoptotic by phosphorylation. We have demonstrated that Bad is phosphorylated on S112 and S136 (Bad-P) in all primary AML and will test the hypothesis that targeting specific kinases known to phosphorylate Bad will induce apoptosis. In support of this hypothesis, the Bad kinases AKT and ERK were found to be expressed and phosphorylated in both, negative AKT on Bad-P and AML cell survival. Preliminary data suggest that Bad dephosphorylation by MAPK/ERK inhibitors and in combination with chemotherapy and retinoids, on Bad-P and apoptosis in vitro with the hope of developing new therapeutic modalities for AML (Specific Aim #2). The factors regulating survival of resistant leukemic cells are not known. In collaboration with Core A (Dr. E. Estey) and Project 2 (Dr. J. Reed), we expect to identify genes that contribute to chemoresistance and relapse by prospectively comparing expression patterns in individual AML patients, at diagnosis and at relapse. Finally, we will test pharmacological targeting of the aforementioned apoptotic pathways in clinical trials (Core A, Dr. E. Estey). We will determine the specific effects of Bcl-2-AS, Bryostatin and Dolastatin on apoptotic pathways and correlate these results with the ability to induce cell death in leukemic cells in patients (Specific Aim #3). In conclusion, we expect to translate the emerging knowledge of the critical roadblocks to apoptosis into novel, molecularly targeted therapeutic strategies for AML.